An animal feed of high digestibility

ABSTRACT

A microbial proteinous substance useful in manufacturing animal feed can be prepared from a hydrocarbon-assimilating yeast of genus Candida by mutating it to mutants by chemical mutagenic agents or physical mutagenic means, inoculating a suspension containing said mutants on a solid culture medium and incubating to form colonies, selecting mutant strains totally or partially deprived of cell wall integrity from cells of said colonies by means of Gram stain and microscopic inspection, proliferating said mutant strains in a culture medium, and utilizing the proliferated cell mass as raw material.

United States Patent Nagasawa et a1.

[ AN ANIMAL FEED OF HIGH DIGESTIBILITY [75] Inventors: Taro Nagasawa, Tokyo; Joji Ono,

Chiba; Tutomu Kudo, Kawasaki; Yoshitugu Harada, Tokyo. all of Japan [73] Assignee: Morinaga Milk Industry Co. Ltd.,

Japan [22] Filed: July 2, 1973 [21} Appl. No.: 375,898

Related U.S. Application Data [62] Division of Ser, No. 142,530, May 21, 1971.

[52] US. Cl. 126/656 [51] Int. Cl A23k 1/00 [58] Field of Search 195/28 R, 34; 426/190, 426/204, 56, 60, 212

[56] References Cited UNITED STATES PATENTS 2,919,195 12/1959 Block 99/105 1 June 10, 1975 3,355,296 11/1967 Perkins et a1 99/14 3,662,672 5/1972 Hoer 99/17 3,679,433 7/1972 Pomeranz et al 99/90 Primary Examiner-Lionel M. Shapiro Assistant Examiner-R. B. Penland Attorney, Agent, or FirmOtto John Munz [5 7 ABSTRACT A microbial proteinous substance useful in manufacturing animal feed can be prepared from a hydrocarbon-assimilating yeast of genus Candida by mutating it to mutants by chemical mutagenic agents or physical mutagenic means, inoculating a suspension containing said mutants on a solid culture medium and incubating to form colonies, selecting mutant strains totally or partially deprived of cell wall integrity from cells of said colonies by means of Gram stain and microscopic inspection, proliferating said mutant strains in a culture medium, and utilizing the proliferated cell mass as raw material.

2 Claims, 4 Drawing Figures PATENTED 3,889,006

SHEET 1 Conrrol 1 Control 1H PATENTEBJUH 10 1915 3.889 .006 sum 2 s wwam; 1 0 05 3,889,006 SHEET 3 OF PROTEIN DIGESTED .b O 8 INCUBATION TIME IN Hrs ANIMAL FEED OF HIGH DIGESTIBILITY This is a divisional application to copending application Ser. No. l42,530. filed May l2, l97l.

The present invention relates to a novel and useful mutant of hydrocarbon-assimilating yeast of genus Candida, a method of obtaining such mutant, a cell obtained by incubating such mutant and an intracellular substance extracted from such cell as well as foods and nutritive medicaments made by utilizing such cells or intracellular substances and manufacturing method thereof.

More particularly, the present invention relates to a Gramnegative mutant obtained by treating a hydrocarbon-assimilating yeast of genus candida using a chemical reagent or a physical means which is high in digestibility, can easily isolate its cell constituents therefrom, substantially deficient in mannan and is deprived of its cell wall integrity, and a Gram-positive mutant which is lower in mannan content than its parent strain and is partially deprived of its cell wall integrity; a method for obtaining such mutant; a cell obtained by inoculating and incubating such mutant on a YM medium or a culture medium containing sulfite pulp waste liquor, hydrocarbon or waste molasses as a main carbon source and, after proliferating, separating the cell, and an intracellular substance obtained by treating such cell chemically or physically such as alkaline treatment or exposure to sonic oscillation; and a method of obtaining such cell or intracellular substance; as well as a method for manufacturing foods, feeds or nutritive medicaments by utilizing these cells or intracellular substances.

Heretofore, the yeast cell has been utilized as food, feed and medicament because of being high in protein and nucleic acid content and containing a large quantity of nutrient such as vitamin. Taming of characteristic yeast strain, in order for adaptation to medium employed, has been tried for a long time. However, such taming has been carried out only in a viewpoint of the ecomization of the production process of yeast cell such as an efficiency of assimilation of carbon source in culture medium, a yield of cell mass, a rate of proliferation, etc., but, as things are, the isolation and induction of strain and mutant which are high in nutritive value in a viewpoint of digestibility for food and feed are not at all carried out. The fact that the yeast cell has a very rigid cell wall around the cytoplasm and the characteristic of this cell wall is due to a complicated higher structure substantially composed of polysaccharides such as mannan, glucan, etc., (D. H. Northcote, Biochemical Journal 5 l, 232, 1952, and P. A. Roelfsen, Biochimica et Biophysica Acta, 10. 477, l953) is evident from the experimental results that, if the yeast cell is treated with a polysaccharide-hydrolysing enzyme produced by, for example, a snail or some microorganisms, the structure of cell wall is disintegrated and is transformed to the protoplast structure in which the cytoplasm or cell membrane is exposed (S. Sugawara, Nature, 212, 92, 1966; G. S. Kobayashi et al, Journal of Bacteriology, 88, 795, 1964; R. E. Domanski, Journal of Bacteriology, 96, 270, 1968; and J. Monreal, Journal of Bacteriology, 94, 24l. I967).

However, yeast cell is little valuable as food and feed since it is very low in digestibility when it is administered to human and animals per se in the intact state (Journal of Japanese Society of Food and Nutrition 23,

62. l970), because a polysaccharide-hydrolyzing enzyme having such property is not found in the digestive fluid of higher animals. This is the main reason for that the application of yeast cells to food or feed industry is retarded.

Much effort has been performed on removal of yeast cell wall, which enable to elevate the digestibility or extractability of intracellular substance in yeast cell. Articles or patent application has been presented on employment ofchemical agents, biological means and mechanical disrupture by means of alkali or enzyme treatment, autolysis, plasmolysis, sonic oscillation, homoge nization and so on. However, these treatment and means are too complicated in operation and to be ex pensive in equipment to be applied in industry.

Therefore, it is desirable in a viewpoint of yeast utilization to induce and isolate a yeast mutant which is deprived of its cell wall integrity totally or partially, and is high in digestibility or isolation efficiency of cellular proteinous substances.

An object of the present invention is to obtain a yeast mutant which is higher in cell digestibility when ingested by human or animals and the intracellular substance thereof is easier in isolation of its intracellular substance and is higher in industrial utility than the conventional yeast cell.

Another object of the present invention is to provide a cell obtained by industrially proliferating the yeast mutant, which is high in digestibility and suitable as a raw material for foods and feeds for human and animal use.

Further another object of the present invention is to isolate an intracellular substance suitable as a raw material for food, feed and nutritive medicaments very efficiently and economically under a mild condition for industrial processing of such cell.

As the result of research for obtaining a mutant which is high in digestibility, easily capable of isolating its intracellular substances and is deprived of its cell wall integrity, from various yeast strains of Family Cryptococcaceae including strains which is capable of assimilating inexpensive carbon source from such a point of view, the present inventors have found that mutants having the desired characteristic can be in duced and isolated.

The mutants of the present invention can be obtained by the following procedure:

A hydrocarbon-assimilating yeast of genus Candida is treated with a chemical mutagenic agent or by a physical mutagenic means to induce mutants; a cell suspension containing the mutants is incubated on a suitable solid medium to form isolated colonies; colonies having a rough surface layer are selected; cells in the colonies are first inspected by an optical microscope to select cells of circular form; and the circular cells are Gram-stained to select a Gram-negative cell. Gramnegative cells are then subjected to chemical determination of mannan content to isolate the mutant cell in which the mannan content is around nil. This is the mutant cell which is deprived of its cell wall integrity and does not contain mannan. On the other hand, cells in the isolated colonies are Gram-stained to select a Gram-positive cell which is low in electron density of outer layer of cell wall by electron microscopic inspection. Mannan content of the cells thus selected are determined and a cell in which mannan content is less than that of parent strain is selected. This is the cell which is partially deprived of its cell wall integrity.

The reason for selecting the mutant of circular form and further isolating a Gram-negative strain is based on the consideration of the present inventors that a yeast cell which is deprived of its cell wall integrity forms a sphere so as to minimize its surface area because it cannot tolerate against the outer pressure otherwise. and on the finding that the site of fixation of primary stain ing by crystal violet at the time of Gram staining is cell wall. that is. discharge of crystal violet fixed on cell wall structure is remarkably delayed when stained cells are rinsed with an organic solvent after staining. (Bacteriological Review, lb; 1, 1952, Stain Technology, 40; 79, 1965 and The Microbial World; second Edition, p. 169, Prentice-Hall Inc., Englewood Cliffs. NJ. U.S.A.) And also a mutant which is modified in structure of cell wall and is partially deprived of its cell wall integrity, even though it is Gram-positive, can be easily detected by inspection by means of an election microscope. The cell which is low in electron density of outer layer of cell wall by the electron microscopic inspection is such a cell that the cell wall structure is modified. the mannan content which is a main ingredient of cell wall is lowered and the cell wall integrity is partially deprived of.

The principle of the present invention consists in ob taining such a mutant strain that its intracellular sub stances can be effectively utilized and easily extracted by applying the above described method to a hydrocarbon-assimilating yeast of genus Candida to obtain a mutant which is totally or partially deprived of its cell wall integrity, in proliferating such mutant, and in utilizing an useful intracellular ingredient in such cell.

FIG. 1 shows micrographs of Gram stained cells of M-7002, M-7005, M-7 and Candida albicans A.T.C.C. I025);

FIG. 2 shows electron micrographs of cells M-7002, M7007 and M-7;

FIG. 3 shows digestion rate of cellular protein of Groups A and 8. Control and CA strains by bovine gastric pepsine as a function of digestion period; and

FIG. 4 shows digestion rate of cellular protein of Groups A and B. Control and CA strains by proteolytic enzyme prepared from pancreas of yellow fish as a function of digestion period.

The present invention is first to expose the cells of hydrccarbon-assimilating yeast of genus Candida to chemical mutagenic agents or physical mutagenic means. As a chemical mutagenic agents. N methyl-N'- nitro-N-nitrosoguanidine, nitrous acid, acriflavin, 4- nitroquinoline-N-oxide. and ethylmethanesulfonate may be used. And as a physical mutagenic means, irradiation by ultraviolet light, irradiation by X-ray, and in radiation by ionizing radiations may be used.

The method of inducing and isolating a mutant in the present invention may be exemplified as follows:

1. Obtaining of Mutant by N-Methyl-N'l litro-N-Nitro Soguanidine According to Lodders text (J. Lodder and N. J. W. KrcgenVan Rij: The Yeast" second Edition. p. 370. North-Holland Publishing Company. Amsterdam. I967 yeast strains of genus Candida was isolated from soil ofJapan, and cells of M-7 strain. one strain of Candida species thus isolated, was inoculated and incubated in YM-medium of pH 6 (0.3% of malt extract. 0.3% of yeast extract, 0.5% peptone. and 1% of glucose in composition) to exponential growth phase, and cells were harvested and resuspended in YM-medium to a cell density of l /ml. After exposing the cell suspen' sion to N-methyl-N'-nitro-N-nitrosoguanidine (K & K

Laboratories Inc.. California. U.S.A.) to a concentration of 40 ug/ml. at 30C for 6 hours. it was diluted with a sterilized saline by 100 times. The diluted suspension was incubated to form isolated colonies on the YM- agar plate prepared by adding 2'/( of agar to the above described YM-medium. Colonies exhibiting a rough 1) surface were selected and then cells in these colonies were taken out and inspected by means of an optical microscope to isolate about 200 strains having a circular form of cell. Each strain was subjected to Gram stain, and three Gram-negative strains were isolated and named as Candida periphelosum M-7002, M-7003 and M-7004, respectively.

The remaining strains were, after treating the cells with glutaraldehyde, fixed with osmium tetraoxide according to the conventional method, or were stained and fixed with perimental Cell Research, 20. 28, I960),

and then were inspected by means of an electron microscope according to Moores procedure (Journal of Biochemical Cytology 3, 225, I957).

Ten strains in which the cell wall, particularly its outer layer is low in electron density, that is, the intensity of blackening on photographic print is low, were separated and further each strain was proliferated in the above mentioned YM medium at 37C for 48 hours and cells were harvested, and, after lyophilysing, the

30 quantities of mannan and glucan in them were determined according to W. E. Trevelyans method (Biochemical Journal. 63, 23, 1956).

A mutant, the mannan content of which is reduced to below 2 g per I00 g of dried cell, was detected and 2 strains complying with this criteria were isolated and named as Candida periphelosum M-7007 and M-7008, respectively.

2. Obtaining of Mutant by Irradiation of Ultraviolet Light 40 Cells of Candida lipolytica NRRL-Y-6795 in expo nential phase of growth were harvested and suspended in a sterilized saline to a cell density of 10 per l ml.

One ml. of this suspension was placed in a petri dish of 9 cm in diameter and, after irradiating at a distance of 30 cm from 15 Watt germicidal lamp manufactured by Toshiba Co.. Ltd. for 3 minutes, was diluted with a sterilized saline by 100 times.

The diluted suspension was incubated on the above mentioned YM-agar plate to form isolated colonies. In the same method as described in l. colonies exhibiting a rough surface were selected and further about 200 strains having a circular form of cell and exhibiting an authentic Gram-negative were isolated therefrom. Each strain was subjected to chemical determination of mannan content according to the procedure by Trevelyan described above. One strain, of which mannan content is substantially nil. was isolated from these strain and named as Candida lipolytica M-7005. This procedure for selection of mannan deficient strain, by chemical determination, is required in the case of the mutant isolation from Candida lipolytica for security because this parent strain originally exhibits an intermediary performance of positive and negative in Gram stain causing obscurity in recognizing Gram-negative mutants. This procedure can be omitted in the case of mutant isolation from other species of Candida since they are authentic Gram-positive organism.

And further from the remaining strains a mutant which is decreased in mannan content to less than 7071 of the parent strain, was detected through the procedures as described in l.

One mutant was isolated complying with above criteria and was named as Candida lipolytica M-7009.

3. Obtaining of Mutant by Nitrous Acid Cells of Candida guilliermondii lFO-l062 which were proliferated in the above described YM medium to log phase of growth were collected and suspended in 0.l m acetate buffer of pH 4.5 containing 0.05 m sodium nitrite to a cell density of per 1 ml.

After allowed to stand at C for minutes, cells were collected by centrifugation and, after washing, were suspended in and diluted with a sterilized solution containing 1% of peptone and 5% of NaCl, by weight per volume, by lOO times.

The diluted suspension was incubated on the YM- agar plate to form colonies. Next, through the same procedure as described in 1, about 300 strains having circular form of cell were separated from colonies of rough surface and one strain of Gram-negative mutant was isolated therefrom and named as Candida guilliermondii M-7006.

4. Obtaining of Mutant by Acriflavin Cells of Candida guilliermondii lFO-l062 was suspended in YM-medium to a cell density of [0 per 1 ml, and acriflavin was added to a concentration of 4 pg/ml thereto and incubated at 30C for 10 hours. Cells were collected and, after washing with a sterilized saline, were diluted with lOO folds volume of the saline.

The diluted suspension was subjected to colony formation on YM-agar plate. One of mutant strain which is lower in mannan content than the parent strain was detected through the same method as described in l and named Candida guilliermondii M-l0.

Nine strains of yeast mutant of genus Candida obtained by the above methods possess an ability of assimilating normal paraffine, and. among these, M- 7002, M-7003, M-7004, M-7005 and M-7006 (hereinafter referred to Group A) are rough in colony surface, circular in cell form and Gram-negative while M-7007. M-7008, and M-70l0 (hereinafter referred to Group B) are smooth in colony surface similarly as in the wild strain and Gram-positive. M-7009 revealed a similar property in colony surface as M-7007, M-7008 and M- 7010, however, it possessed an intermediary perform ance of Gram-positive and Gram-negative.

The microbiological properties of these yeast mutant of genus Candida are as shown in Table 1. Among these strains M-7002, M-7005, M-7006, M-7007 and M- 70l0 are tabulated hereinbelow along with their corresponding A.T.C.C. registration numbers, which were secured by depositing samples with the American Type Culture Collection in Washington, D. C. on February 24, I971.

Microorganism name A.T.C.C. number Candida albicans A.T.C.C. 10259 as a typical example of conventional wild strain were incubated on the above described YM-agar plate at 30C for 3 days to obtain isolated colonies.

It was found that the colony surface of M-7002 was rough while those of M-7007 and Candida alhicuns were smooth.

Table l Microbiological properties of mutants Group A Group B (M-7002-M-70(l6l (M7007-M-7(ll0) glucose galactose Assimilation lactose of organic maltose carbon sucrose compounds ethanol n-paraft'me peptone Utilization urea z of nitrogen NH,Cl compounds KNO;

Biotin lnosine Nicotic acid Vitamine Pantothenic acid requirment Pyridoxin Thiamine pAminobenzoic acid Gram stain ne ative positive" Cell form sp ere rod Cell dimension 5 o p. 3 u Colony surface rough smooth Range of growth temp. ll 37C 0 37C Protein content 5'9 64 59 9;

' M-TUUQ is exceptional in exhibiting (irum r Other strains of Group A. other strains of Group B M-7002 and M-7005 are Gram-negative while Control and the other conventional wild strains comprising (l) and Control (III) are Gram-positive. The perform- Candi'a'u lipolyrir'a. Candida guillicrmmrdii and Candida ance of M 7003, M-7004. M-7005 against Gram Slainurz'lz's. etcv were similar as M-7002. M-7007 and Caning was quite identical with that of M-7002 and they dida albicans, respectively. in feature of their colony 5 e concluded to be Gram-negative M-7007. M- Suffacfl 7008. and M-7010 were, on the contrary. responded The mutants of group A among the? mulams of against the staining in the same mode as the convengenus Candida. a remarkable difference from tinnal wild strain unyincing that these are Gram the conventional wild strains such as M7 (Control l positive. M-7009 was concluded to be intermediary of C(Ifldl dfl guilliermoudii ill'l authentic exam- Grun positive and Grarnqqegative Next Cells fg mu- P of hydrocarbohfissh'hhating Yeast (Control (In) tants. 3 control strains and 2 CA strains were collected and Candida albicans A.T.C.C. 10259 (Control (lll in and, after hi l hili a d then their mannan that the mutants are Gram-negative and contain no and glucan Contents were d r i d d according mllhhahto the above described Trevelyan method.

while mutants f group B are lower in manna And also the relative content of mannan in mutants tent than the conventional wild strains in spite of being were Calculated based on 100% f th mar-man content similarly positive in Gram stain as the control strainsv in comm] strain. Th results are h n i Table 2. M-7009 is exceptional in exhibiting an intermediary From Table 2 it was proved h mutants 54-7002 M- performance of positive and negative in Gram stain. 7003, MI/Q04 54-7005 d M-7006, that is Group A Evidences for above Conclusion are Pmsemed in the strains. are remarkably distinguished from the control following. The above described [2 strains and Candida Strains in two respects h t h e Gram-negative and lipolica NRRL-67 5 and Candida t piw s lAM-4l47 mannan content in them is substantially nil while M- (these 2 strains are hereinafter abbreviated to CA 7()()7 M-7008, and M-70l0, that is Group B strains.

S l4 sll'flihs in tOlflL were ihhlfbated y ki g are Gram-positive like as the control strains but their Cuhure medium of P colhpnslhg a COmPOSmOh mannan contents are decreased to 7 l .410.7% of that of WC of normal pal'affihe havlhg 14 to 18 carbon ofthe control. Candidalipolytica and M-7009 were only atom 05% 0f NHGCI, (17% 0f z u 002% Of exception in exhibiting an intermediary response t- 2 Of 4- 2 001% Of 2- against Gram stain. and the mannan content in the lat- H 0 and 0.05% of yeast extract at C for 48 hours. {er w i ed within th ran e of Group B,

Cells were collected by centrifugation and a portion 30 A cordi to th ex eriments carried out by the thereof was Gram stained and inspected by a micro present inventors. the mannan contents of other three Sc pe h C g P (magflificfltiofll 0), m conventional strains that is Candida pampsilosis. Cantants M-7002 and M-7005 as typical examples and didainrermedia and Candida robusla,proliferated under Control (I) and Control (Ill) after Gram staining are the above incubation conditions were high value of 3.0

shown in FIG. 1. Violet-colored cells show that crystal i 0.2%.

Table 2 Polysaccharides Content Relative Gram stain (g/ lOOg dried cells) content of mannan glucan mannnn Control I!) 7.2 2.8

Control ill) 7.0 2.8 (IOU) Control llll) 6.7 3.0

M4004 6.6 u t) M-70o6 6.4 0 o M-7008 6.6 l. I 39.3

Candida Iiprdyriru 2 9 l ll 7 2 NRRL-6795 Candida lmpitulis t 9.3 2.3 81.2

violet was fixed on cell wall after primary staining and And also, in Table 2. the relative content of mannan are recognized to be Gram-positive while green colored in IWO A trains Sho n in the bottom column werem cells are such cells that crystal violet was not fixed but In Spite of e the Conventional Wild Strains. and eluted out by organic solvent treatment and reacted 822%, p yt of the 60mm] Strains which a with secondary taining methyl green and are re glOW avlues equivalent 0 that Of some mutants in the nized to be Gram-negative. From FIG. 1, it is clear that Prewm lhvemloh- The expel'lmehtal results shQwihg that these CA strains are lower in mannan content than other conventional wild strains was presented by Nabeshima et al. (Journal of Technology 48. 556, I970). However, according to the observation by means of an electron microscope. these CA strains are high in electron density of cell wall and kept remained their cell wall integrity. Therefore, a favorable effect observed in the mutants ofthe present invention as described below can not be obtained in CA strains.

Next. cells of M-7002, M7007. and M-7 strains as representatives of the mutants of the above described Groups A, B and the control strain, respectively. were observed by means of an electron micrograph (magnifh cation 20,000) according to the method described in (l The results are as shown in FIG. 2.

The control and M-7007 strains are observed to possess a distinct cell wall in peripheral area of cells, while the thickness of cell wall portion is thin and the electron density is low in M-7002 strain. That is, it is clear that M-7002 strain is deprived of its cell wall integrity and that it is about twice the control strain in cell width. And also. according to the observation by an electron microscope, it is clear that M-7003, M-7004, M-7005, and M 7006 are similarly deprived of their cell wall integrity. As already described, from facts that the site of primary staining by crystal violet is a cell wall and M- 7002, M-7003. M-7004, M-7005, and M-7006 are Gram-negative, that the main component of cell wall is mannan and the mutants of the present invention do not contain mannan or are lower in mannan content than the native strain, and that M-7002, M-7003, M- 7004, M-7005, and M-7006 reveal defective cell wall structure by electron microscope observation, it was confirmed that M-7002, M-7003, M-7004. M-7005, and M-7006, that is, Group A mutants are deprived of cell wall integrity while M-7007, M-7008, M-7009, and M-70l0, that is Group B mutants are partially deprived of cell wall integrity. From the fact that cell wall lowers the digestibility and the utilization efficiency of intra cellular substances of yeast cell. the above described mutants of the present invention are remarkably excellent in both digestibility and utilization of intracellular protein in comparison with the conventional strain. Next, experiments were conducted in order to provide the evidences to show the favorable effect of mutants of the present invention in digestibility and protein extractability out of cells. The results are as follows:

In the first experiment. cellular proteins of 9 strains of the above described mutants of genus Candida, 3 strains of the control and 2 CA strains were tested on their digestibilities by bovine gastric pepsin and proteolytic enzyme prepared from pancreas of a yellow-tail fish. These 14 yeast strains were respectively incubated by shaking in the normal-paraffine medium above described, at 30C for 48 hours and proliferated cells were harvested by centrifugation. Then after washed and dried, each cell mass was suspended in hydrochloric acid of pH L8 to 1% cell density. Bovine gastric pepsin (2 X crystallized and lyophilized, l53l370-l, manufactured by Sigma Chemical Company) was added to each suspension to 0.571 by weight per volume, and the digestion was allowed at 37C. Portions of reaction mixture were pipetted at the reaction periods indicated in FIG. 3. The centrifugation was carried out, and the nitrogen content in supernatant fluid was determinded in accordance with Kjeldahl method. The digestion rate of protein, expressed in 7c of the above nitrogen content to the total nitrogen content in the cell mass before digestion, was similarly determined. The results are shown in FIG. 3.

In FIG. 3, the ordinate designates the digestibility of intracellular protein of yeast by bovine gastric pepsin in and the abscissa designates the digestion time in hours. Lines A, B. C. and CA show average digestibilities of Group A mutants, Group B mutants. control strains, and CA strains respectively. As shown in FIG. 3, Group A mutants are higher about 2.7 times the conventional wild strain in average digestibility when comparing in 24 hours after digestion commences and the average digestibility of Group B mutants is about l.o times that of the conventional wild strain. In addition. the digestibility in the mutants of the present invention increases even at 20 hours after the digesion started while that in the conventional strain reaches to plateau in l0 hours after the digestion started Therefore. the yeast mutants isolated by the present inventors was proved to be very easily digested. Next, the digestion rate of cells of the mutants was tested by proteolytic enzyme of fish origin in order to confirm the digestibility of them in case of using them as feed for fish cultivation. The cells were suspended in 0.lM-borate buffer (pH 8.0) to 1% cell density and proteolytic enzyme, prepared from pancreas of a yellow-tail fish according to Laskowski method (Methods of Enzymology, Vol. II, page 8, first Ed; edited by S. P. Colowick and N. 0. Kaplan, Academic Press Inc., New York, I955), and 0005M CaCl were added to the suspension to a concentration of 0.5% by weight per volume. The digestion was allowed at 37C, and the digestibility was determined in the same method as employed in digestibility determination by bovine gastric pepsin. The results are shown in FIG, 4. In FIG. 4, the ordinate indicates the digestibility of cellular protein of yeast by proteolytic enzyme prepared from pancreas of a yellow-tail fish, and the abscissa indicates the digestion time. The digestibilities of Group A mutants, Group B mutants, control strains and CA strains are presented by curves A, B, C, and CA, respectively. Group A and Group B mutants were respectively 2.2 and 1.8 times higher the control strains in digestibility when digested with enzyme of pancreas of a yellow-tail fish. Therefore the mutants of the present invention, particularly Group A mutants, were remarkably improved in digestibility in comparison with the control strains. And, even with any of these enzyme systems, CA strains were comparable to the control strains in digestibility. As shown in Table 2, although CA strains are slightly lower in mannan content than the control strains, CA strains are remarkably inferior to the mutants of the present invention in digestibility of cellular protein. CA strains are not subjected to a treatment for mutation induction according to the method of the present invention and are high in electron density in peripheral area of cells when observed by an electron microscope, and therefore, are considered to be not deprived of their cell wall integrities. Accordingly, differing from the control strains and CA strains, the cellular protein of mutants of the present invention are in such a state as being easily digested, because the mutants of the present invention are not only deficient in mannan or decreased in mannan content but also their cytoplasm or cell membrane is directly exposed to the medium since the cell wall integrity is totally or partially deprived.

Next, the efficiency of extracting the intra-cellular substances from these mutants. control strains and CA strains by chemical reagents or physical means was determined for the purpose of design of devices for extracting and isolating intra cellular substances.

Table 3 Protein extraction out of cells 91' of protein extracted Through the same procedure as in the case of experiments shown in FIGS. 3 and 4, nine strains of the above described mutants, 3 control strains and 2 CA strains were proliferated in normal paraffine medium. Cells were collected and suspended in 0.05N-NaOI-l solution in 1% of cell density and heated at 50C for 2 hours and, after centrifugating, nitrogen content in supernatant fraction was determined according to Kjeldahl method, and the extraction rate (70) was calculated from the ratio of the amount of nitrogen extracted in NaOH solution to the total amount of nitrogen in the cell before treatment. The result is shown in column 2 of Table 3. Cells of the above described three groups were respectively suspended in a sterilized saline to 5% of cell density and 5 ml portions of the suspensions were disrupted by means of a lOKC sonic disintegrator for 5 minutes, thereafter centrifugated, and nitrogen contentent in super-natant fraction was determined. The extraction rate (70) was calculated in the same manner and the result is shown in column 3 of Table 3.

As shown in Table 3, it is clear that the mutants obtained in the present invention are remarkably higher in protein extraction rate by alkaline solution treatment and sonic oscillation in comparison with control strains and, similarly as in the digestibility test by enzyme, the protein extraction rate is elevated as a function of decrease in the mannan content. in spite of lower mannan content in CA strains, rate of protein extraction from them was remained to the similar level as that from the conventional strains. As described in the experimental results shown in FIGS. 3 and 4, it is also clear that the mutants obtained according to the method of the present invention not only possess an unique property to be deprived of mannan or to be lower in mannan content in comparison with the control strains but also to be characterized by genetically defected cell wall structure, resulted in remarkable improvement in protein extraction rate by alkaline treatment or sonic oscillation in comparison with control strains and CA strains. In addition, the mutants of the present invention are su perior to the conventional wild strains in extraction efficiency of intracellular substances in cases of urea treatment, autolysis, mechanical grinding, and freeze and thaw treatments. From a fact that M-7002, M-

7003, M 1004, M-7005, and M-7006 strains among yeast mutants of genus Candida isolated in the present invention are Gram-negative while all conventional yeast strains are Gram-positive regardless of their growth condition and growth phase, an information that the Gram straining is a method for discriminating a cell wall, an observation by an electron microscope and an experimental result that the digestibility by enzyme and the rate of protein extraction are high, these yeast mutants of genus Candida in the present invention, being different from the conventional strains, has such a characteristics that the cell wall integrity is deprived and the cytoplasm or cell membrane is exposed. It is also clear that four mutants, M-7007, M-7008, and M-l0 are Gram-positive similarly as the conventional strain of yeast incubated under the same condition and M7009 is intermediary of Gram-positive and Gram-negative, but their mannan contents are decreased to 70 to 10% of that of the control strains of genus Candida, From a fact that the effect such as rate of enzymatic digestion or extraction of protein, is in an intermediate degree of the above described mutants deprived of cell wall integrity and the control strain, these four mutants are presumed to be partially deprived of their cell wall integrity. Among the conventional strains of yeast of genus Candida, Candida lipolytica and Candida tropiculis (CA strains) are slightly lower in mannan content than the control strains and Candida lipolytica possesses an intermediary property of Gram-positive and Gram-negative. However these CA strains are remarkably lower in rate of protein digested and in extraction rate than the mutants of the present invention, and, therefore, these CA strains quite differ from the mutants of the present invention.

The yeast mutants of genus Candida of the present invention isolated as descrived above are such a mutant as deprived of cell wall integrity and having a feature of being Gram-negative and not containing mannan, or as being Gram-positive but partially having lost mannan, and are, being different from the usual conventional wild strains of yeast, high in digestibility by a digestive enzyme and are very easy in extraction of intracellular protein by various treatments. Therefore, in case of using yeast cells for human food and feed for domestic animals or fishes, the yeast mutants of genus Candida of the present invention provide highly digestive product, and it is also possible to manufacture a nutritive medicament having a very high nutritive value. Whereas, in case of industrially isolating and separating intracellular substances of yeast, the cost of equipment and expenses for processing can be reduced when the yeast mutants of the present invention are employed, and also the decomposition and denaturation of product can be prevented since the isolation and separation of intracellular substances and autolysis can be carried out under mild conditions. Also, in a viewpoint of extraction and utilization of a biologically active substance such as enzyme, the method of the present invention can be employed with remarkable advantage in comparison with the usual procedure.

EXAMPLE 1 Twenty liters of culture medium (pH 6.0) comprising a composition of, by weight, 5% (as carbohydrate concentration) of molasses for fermentation, 0.25 7c of (NH SO,, 0.5% of KH PO 0.02% of MgSO,. 7H,O, and 0.04% of yeast extract were, after boiling, filtered and sterilized. The mutant M-7007 was inoculated thereto and incubated during aeration at 30C for 24 hours. This was added to 500 I of the above described sterilized culture medium and incubated during aeration at 30C for 36 hours and then at 40C incubated during aeration at 30C for 36 hours and then at 40C for 12 hours. Cells were collected by centrifugation and, after washing with water. dried in a drum dryer to obtain 3 kg of dried yeast cell. Two kg were taken out therefrom, added with 20 kg of 0.1N NaOH and, after allowed to stand at 50C for 2 hours. centrifugated. The supernatant was adjusted pH to 4.8 by adding 1N HCl to precipitate protein which was then separated by centrifugation and dried by means of a small spray dryer to obtain 500 g of dried yeast intracellular protein. Fifty kg of water and 1 kg of glucose were added to 500 g of the protein. The fluid was subjected to lactic acid fermentation by incubating with Lactobacillus acidophilus at 37C for 30 hours and added with sugar and flavor to obtain a palatable and nutritive protein-rich beverage.

EXAMPLE 2 The mutant M-7006 was inoculated to 20 l of culture medium (pH 6.0) comprising a composition of, by weight, 4% (as carbohydrate concentration) of desulfited sulfite-pulp liquor, 0.2% of (NH SO 0.1% of KH PO 0.25% of MgSO .7H O and 0.3% of yeast extract. After incubated at 30C for 48 hours, the fluid was added to 500 l of the above described sterilized culture medium and incubated during aeration at 30C for 48 hours. Cells were harvested by centrifugation and, after washing with water, were dried in a drum dryer to obtain 2.5 kg of dried yeast cell, which were then powdered by means of a grinder to obtain a nutritive medicament which is high in protein and vitamin content and high in digestibility.

One kg of yeast cell obtained as above was mixed with 5 kg of wheat flour, 40 g of salt, g of honey and 2 kg of water and further added with 3.5 kg of gluten, 300 g of edible oil and flavour. The mixture was then baked in an oven at 120C for 30 minutes to obtain a crisp snack food for diet which possesses a balanced amino acid composition.

EXAMPLE 3 Twenty liters of culture medium (pH 5.5) comprising a composition of 1% of normal paraffine having 14 to 18 carbon atoms. 0.5% of NH Cl, 0.7% of KH PO 0.001% of FeSO,.7H 0. 0.02% of MgSO,.7H O. 0.001% of MnSO,.7H O, 0.01% of CaCl .2H-,O and 0.05% of yeast extract, by weight, were sterilized. And then the mutant M-7002 was inoculated thereto and incubated during aeration at 30C for 24 hours. Thereafter. this culture medium was added to 500 l of the sterilized culture medium above described. and further incubated during aeration at 30C for 48 hours while adjusting pH and then cells were collected by centrifugation dryer to obtain 2.5 kg of dried yeast cell having high digestibility. Two kg of the dried cell mass were taken out therefrom and 12 kg of wheat flour were added thereto. After kneading, the mixture was blended with 6 kg of white fish meal to obtain feed for fishes.

Four kg of dried yeast cell prepared according to the same method as above were added to 20 kg of 0.5N NaOH and, after allowed to stand at 50C for 2 hours, centrifuged to separate the supernatant containing intracellular substances. Two kg of CaCl .2H O and 0.2 kg of Ca(OH) were suspended in 15 kg of water at 90C, and then the above supernatant was added thereto while stirring. After allowed to stand for 30 minutes, precipitate was separated by centrifugation, washed with and then was suspended in water, and adjusted pH to approximately 7 by adding 6N HCl. The precipitate was centrifuged to obtain a flake-like and meat-like textured foodstuff.

What is claimed is:

1. A foodstuff of high digestibility containing a yeast mass of cells obtained by 1 alkaline treating (2) precipitating and (3) drying of yeast cells which are at least partially deprived of cell wall integrity and at least partially devoid of mannan, said yeast cells being selected from the group consisting of hydrocarbon assimilating mutants of Candida MA7002, M7005, M7006, M7007 and M7010.

2. A foodstuff as claimed in claim 1, said cells being (C Gram positive cells of low electron density of the outer region of their walls, the mannan contents of said cells being below of the mannan content of the parent strains. 

1. A FOODSTUFF OF HIGH DISESTIBILITY CONTAINING A YEAST MASS OF CELLS OBTAINED BY (1) ALKALINE TREATING (2) PRECIPITATING AND (3) DRYING OF YEAST CELLS WHICH ARE AT LEAST PARTIALLY DEPRIVED OF CELL WALL INTEGRITY AND AT LEAST PARTIALLY DEVIOD OF MANNAN, SAID YEAST CELLS BEING SELECTED FROM THE GROUP CONSISTING OF HYDROCARBON ASSIMILATING MUTANTS OF CANADA MA7002, M75005, M7006, M7007 AND M7010.
 2. A foodstuff as claimed in claim 1, said cells being (C2) Gram positive cells of low electron density of the outer region of their walls, the mannan contents of said cells being below 70% of the mannan content of the parent strains. 